The functional properties of most proteins are regulated by post-translational modifications (PTM) of specific residues. Up to now, phosphorylation at serine, threonine or tyrosine is the most frequent experimentally determined PTM1 (http://selene.princeton.edu/PTMCuration). In eukaryotes, 30% (S. cerevisiae) to 50% (mouse) of protein species are phosphorylated2,3. Proteins of critical importance may have multiple phosphorylation sites, serving to activate or inactivate a protein, promote its degradation, or modulate interactions with protein partners4. For example, p53 has at least 18 phosphorylation sites4,5. Importantly, multi-site modifications can occur in different combinations, leading to different functional forms of a protein6.
Early studies of protein phosphorylation relied on 2D gel electrophoresis, which is based on changes in protein electrophoretic mobility and isoelectric point caused by the incorporation of phosphate groups7. 2D gel electrophoresis cannot resolve different phosphorylation sites within the same protein8,9. Recently, mass spectrometry (MS) of the phosphoproteome has come to the fore for studies of phosphorylation in vivo. Through the use of protease digestion and high-resolution MS, thousands of phosphoprotein species can be identified9,10. When samples from different sources (e.g. treated and control) are differentially labelled with isotopes, changes in the levels of phosphorylation at specific sites in specific proteins can be estimated11. Despite these advances, the determination of patterns of phosphorylation within individual protein molecules remains challenging2. For example, proteins monophosphorylated on one of two adjacent sites are difficult to distinguish. The occupancy and connectivity of phosphorylation sites is a problem ideally suited for single-molecule approaches.
Engineered protein nanopores have been used for the stochastic detection of a wide variety of molecules in solution12,13, ranging from divalent metal cations14 to organic molecules15 to chemically reactive substances16. Nanopore technology has also been investigated as an ultra-rapid, low-cost platform for single-molecule sequencing of DNA and RNA17,18. For example, single-stranded DNA can be ratcheted through protein pores with enzymes19-22 and the sequence read23-27 from base-dependent transitions in the ionic current28-31.